Cryptococcal meningitis (CM) is a severe disease that affects both HIV and non-HIV infected individuals with almost a quarter of a million deaths annually globally. Despite antiviral treatment of HIV, CM continues to be a problem in the U.S. with about 6000 infections annually. Attributable mortality remains at 30-50% despite therapy and no meaningful new anti-fungal therapies have been developed in the modern era. Our long-term objective is to assess the role of host and pathogen factors in the susceptibility and outcome of cryptococcosis to improve patient outcomes. 1) Role of the RNA-binding protein DDX6 in fungal disease and autoimmunity. Our previous work characterized the role of mammalian DDX6 in the post-transcriptional regulation of autophagy, a process of cellular protein turnover that regulates the inflammasome-associated cytokine IL1B and associated this new regulatory pathway with autoimmunity in a series of patients with PIK3CD/p110 gain-of function mutations. DDX6 recruits mRNA targeted for degradation to a decapping protein, Dcp2 which removes the 5 mRNA cap, potentiating a sequence of degradation steps. Activation of Dcp2 requires phosphorylation by the target of rapamycin mTOR, which is a central regulator of innate and adaptive immunity. Currently, we have developed a specific monoclonal antibody that can be used to quantitate TOR-dependent phosphorylation by a convenient flow-based assay, to assess levels of MTOR activation in patients receiving rapamycin therapy. We are also expanding this work to study the role of granulocyte-monocyte colony stimulating factor (GMCSF) in the regulation of autophagy, IL1B and defense against the fungus, Cryptococcus gattii in concert with our project 2 on immune defects in patients with CM. Characterization of this regulatory process and the generation of specific reagents will be valuable in our studies characterizing susceptibility to C. gattii and the role of our previously described autoantibodies against GMCSF as well as a number of specific mutations discovered in our cohort of previously healthy patients with cryptococcal disease. In addition, we have found that the cryptococcal homolog (Vad1) of mammalian DDX6 also plays a central role in the regulation of fungal virulence. We are currently analyzing several pathways involved in TOR-dependent signaling resulting in the epigenetic regulation of several fungal genes whose transcriptional levels correlate with poor outcome (death) in a cohort of 60 HIV/AIDS infected patients. 2) New drugs for cryptococcal disease. Fungicidal activity of a drug is particularly important in cryptococcal meningitis where fungistatic drugs such as fluconazole have a 100% mortality despite therapy. We are also conducting pre-clinical studies in collaboration with Matinas Biopharma, to develop an orally available cochleate formulation of amphotericin B for CM.